Method of detecting and discriminating between nucleic acid sequences

ABSTRACT

The present invention is directed to a method for detecting the presence or absence of any specific target nucleic acid sequence contained in a sample. The target sequence can be present in the sample in a relatively pure form or as a component of a member of a mixture of different nucleic acids. The method of the invention utilizes a novel primer design. The sequence of the novel primer is composed of two portions, the 3′ portion is a primer specific for the desired nucleic acid sequence and the 5′ portion is complementary to preselected nucleic acid sequence. Extension of the 3′ portion of the primer with a labeled deoxynucleosides triphosphate yields a labeled extension product if, but only if, the template includes the target sequence. The labeled extension product is detected by hybridization of the 5′ portion to the preselected sequence. The preselected sequence is preferably bound to a solid support as one member of a grid having a group of sequences.

[0001] This invention was made with government support under Grant No.HG00099 awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF INVENTION

[0002] The present invention relates to a method for detecting and fordiscriminating between nucleic acid sequences if present in a sample.More specifically, the invention relates to a method for determining ifa particular DNA or RNA sequence is-present in a sample. The inventionalso relates to discriminating between sequences which differ from eachother by as little as a single nucleotide. The DNA or RNA may be singleor double stranded, and may be a single species or a component of amixture of nucleic acids. The method of the invention utilizes a novelprimer design. The sequence of the primer is composed of two portions,the 3′ portion is a primer specific for the target nucleic acid sequenceand the 5′ portion is complementary to a preselected nucleic acidsequence. Extension of the 3′ portion of the primer with labeleddeoxynucleosides triphosphate yields a labeled extension product if, butonly if, the template includes the target sequence. The labeledextension product is detected by hybridization of the 5′ portion to thepreselected sequence.

BACKGROUND OF THE INVENTION

[0003] The genome of an organism is unique. Not only do the genomes ofdifferent species differ, but the genomes of different individualswithin a species differ (with the exception of identical twins orclones) These differences provide individual and species specificcharacteristics which can be used for identification by nucleic acidbiochemical techniques such as hybridization and polymerase mediatedreactions, both dependent for their specificity on precise base pairing.

[0004] The goal of nucleic acid based diagnostics is the detection ofspecific nucleic acid sequences. This goal often requires the detectionof a specific sequence in the presence of other sequences. In certaincases it is necessary-to discriminate between closely related sequences,even sequences which differ by only a single nucleotide. Prior artmethods for doing so are described in various publications. For example,the use of allele-specific oligonucleotide (ASO) hybridization probesfor the detection of specific nucleic acid sequences has been described(Wu et al., DNA 8:135-142 (1989); Thein, et al., Br. J. Haematol.70:225-231 (1988); Connor, et al., Proc. Natl. Acad. Sci. USA 80:278-282(1983); Studencki, et al., Am. J. Hum. Genet. 37:42-51 (1985); Pirastu,et al., N. Engl. J. Med. 309:284-287 (1983); Orkin, et al., J. Clin.Invest. 71:775-779 (1983); Thein and Wallace, The use of syntheticoligonucleotides as specific hybridization probes in the diagnosis ofgenetic disorders. In Human genetic diseases: A practical approach. K.E. Davies, ed. (Oxford; IRL Press), pp. 33-50 (1986)). This approachallows the discrimination between nucleic acids which differ by aslittle as a single nucleotide (e.g., alleles). Individual hybridizationreactions are required for each allele to be detected. Erlich, et al.,Eur. J. Immunogenet. 18:33-55 (1991) and Zhang, et al., Nucleic AcidsRes. 19:3929-3933 (1991) have recently described the use of immobilizedASO probes. In this method, a set of ASO probes is immobilized on amembrane and hybridized with labeled polymerase chain reaction (PCR)products. Under appropriate conditions hybridization is allele specific.Each hybridization can analyze only a single amplification reaction. Thepresent invention allows for the detection of specific sequences in asample. Because the template specific step and the detection step areeach controlled by specific but independent base pairing requirements,the overall process allows detection of multiple templates and multiplesamples simultaneously.

[0005] The concept of in vitro DNA amplification was first proposed byKhorana and coworkers in 1971 (Kleppe, et al., J. Mol. Biol. 56:341-361(1971)). Realizing that total chemical synthesis of a gene would resultin a finite amount of product, a procedure for in vitro replication wasproposed. Their procedure was based on extensive studies of the repairreplication reaction, the in vitro replication of a DNA template using acomplementary primer (Kleppe, supra). Their proposal was as follows:“The DNA duplex would be denatured to form single strands. Thisdenaturation step would be carried out in the presence of a sufficientlylarge excess of the two appropriate primers. Upon cooling, one wouldhope to obtain two structures, each containing the full length of thetemplate strand appropriately complexed with the primer. DNA polymerasewill be added to complete the process of repair replication. Twomolecules of the original duplex should result. The whole cycle could berepeated, there being added every time a fresh dose of the enzyme.” Morerecently, this in vitro amplification process has been further developedinto the polymerase chain reaction (Mullis, et al., Cold Spring HarborSymp. Quant. Biol. 51:263-273 (1986); Saiki, et ale, Science230:1350-1354 (1985); U.S. Patent No. 4,683,202). Although templateamplification improves detection of a particular sequence because alarger amount of template is available for analysis, post amplificationsteps are often required to detect specific sequences within theamplified product. For example, ASO hybridization has been combined withPCR amplification for the specific detection of various disease alleles(Impraim, et al., Biochem. Biophys. Res. Commun. 142:710-716 (1987);Saiki, et al., Nature 324:163-166 (1986); Farr, et al., Proc. Natl.Acad. Sci. USA 85:1629-1633 (1988); Saiki, et al., N. Enql. J. Med.319:537-541 (1988); Chehab, et al., Nature 329:293-294 (1987)). Thepresent invention provides an alternative for the analysis of PCRamplification products to determine the presence or absence of specificsequences.

SUMMARY OF THE INVENTION

[0006] This invention provides a method for determining the presence orabsence of a target nucleic acid sequence present in a sample and fordiscriminating between any two nucleic acid sequences even if suchsequences differ only by a single nucleotide. The nucleic sequences maybe single or double stranded DNA or RNA. The target sequence may berelatively pure species or a component of a nucleic acid mixture. It maybe produced by an in vitro amplification such as a PCR or ligationamplification reaction or by a plurality of cycles of primer extension.The method of the invention entails extension of a novel, two componentprimer on templates which may or may not include a target nucleic acidsequence. The 3′ portion of the primer is complementary to a portion ofthe template adjacent the target sequence. The 5′ portion of the primeris complementary to a different preselected nucleic acid sequence.Extension of the 3′ portion of the primer with a labeled deoxynucleosidetriphosphates yields a labeled extension product if, but only if, thetemplate includes the target sequence. The presence of such a labeledprimer extension product is detected by hybridization of the 5′ portionto the preselected sequence. The preselected sequence is preferablyimmobilized on a solid support. A plurality of preselected sequencesimmobilized on a solid support to provide an array for concurrentscreening of a plurality of labeled primer extension products isprovided.

[0007] One practical embodiment of the invention relates to methods fordiagnosing diseases such as sickle cell anemia or thalassemia caused bya defective allele. Kits for performing such a diagnosis are provided.

DESCRIPTION OF THE FIGURES

[0008]FIG. 1 depicts a two component primer useful in this invention.

[0009]FIG. 2 depicts one embodiment of the invention.

[0010]FIG. 3 depicts a solid support having a plurality of preselectedsequences immobilized in a particular array on a grid.

[0011]FIG. 4 depicts the application of the present invention for thedetection of alleles of the human TYR locus.

[0012]FIG. 5 depicts the genotypimc-analysis of ten individuals foralleles at the TYR locus using the invention.

DEFINITIONS

[0013] As used herein, the following words have the meaning set forth:

[0014] “Oligonucleotide” refers to a nucleic acid sequence composed oftwo or more nucleotides. It can be derived from natural sources but isoften synthesized chemically. It can be of any length. It is generallyused as a primer, a probe or a component of ligation reaction.

[0015] “Primer” refers to an oligonucleotide which is used to initiatenucleic acid synthesis by a template dependent polymerase such as a DNApolymerase. The primer is complementary to a portion of a templatenucleic acid.

[0016] “Primer extension” refers to the process of elongation of aprimer on a nucleic acid template. Using appropriate buffers, pH, saltsand nucleoside triphosphates, a template dependent polymerase such as aDNA polymerase incorporates a nucleotide complementary to the templatestrand on the 3′ end of a primer which is annealed to a template. Thepolymerase will thus synthesize a faithful complementary copy of thetemplate. If only a single nucleoside triphosphate is present in aprimer extension reaction, then that nucleotide will be incorporated inthe primer extension product only if the base of the nucleosidetriphosphate is complementary to the base of the template immediatelyadjacent to the 3′ end of the primer.

[0017] “Allele” refers to one of two or more alternative forms of a geneoccupying corresponding sites (loci) on homologous chromosomes. The DNAsequence of alleles of a locus differ from each other by at least onenucleotide.

[0018] “Target” refers to the nucleic acid sequence to be detected ordiscriminated.

[0019] “Sample” refers to a nucleic acid which may or may not containthe target. The sample nucleic acid may be DNA or RNA, single or doublestranded and present in a relatively pure form in the sample or onecomponent of a mixture in the sample. In the case of RNAsit is oftenuseful to convert the RNA into DNA using a reverse transcription step.The DNA product can then be analyzed directly or subjected to anamplification step prior to analysis by the present invention.

[0020] “Allele specific primer extension (ASPE)” refers to a method asdisclosed in copending application Ser. No. 07/683,137 filed Apr. 10,1991 pursuant to which an oligonucleotide primer is annealed to a DNAtemplate 3, with respect to a nucleotide indicative of the presence orabsence of a target allelic variation. The primer is then extended inthe presence of labelled dNTP in which the N is complementary to thenucleotide indicative of the presence or absence of the target allelicvariation in the template.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The invention is described by reference to the Figures. As shownby FIG. 1, the 3′ portion of the primer is complementary to a sequenceadjacent a target sequence which may or may not be present in a sample.The 5′ portion of the primer is complementary to a known or preselectedsequence preferably immobilized on a solid support and arranged in aparticular pattern. The two components or the primer may nave anydesired number of nucleotides. The number of nucleotides in each portionof the primer may be the same or different. Preferably each portion ofthe primer contains from about 10 to about 100 nucleotides. The word“about” indicates a variance e.g., of plus or minus ten nucleotides.

[0022]FIG. 2 illustrates the principle of the invention as applied toidentify and discriminate between two allelic nucleic acid sequenceswhich may be present in a sample and which differ by a singlenucleotide. As illustrated, a sample may contain either “A-T” or “C-G”alleles.

[0023] The primer is hybridized to the alleles immediately adjacent thevariant nucleotides responsible for the allelism. ASPE reactions areperformed with each of four labeled deoxynucleoside triphosphatesindependently only one of the primer extension reactions will label theprimer for each of the different alleles.

[0024] As specifically depicted by FIG. 2, the amplification product issubjected to hybridization with a primer which includes a 3′ portioncomplementary to the “A-T” allele which is then subjected to extensionwith labeled dNTP. Extension will occur only with dTTP if the A-T alleleis present in the sample. No extension occurs with-ATP, dCTP or dGTP.

[0025] The labeled primer extension product is screened by hybridizationof the 5′ portion of the labeled primer with the solid support depictedby FIG. 3. Each location on the array of sequences contains a differentpreselected oligonucleotide. The solid support may be of any size withany desired number of locations. The preselected oligonucleotidesimmobilized at each of the grid locations are preferably at least10-100, preferably 15 to 25 nucleotides in length. Hybridizationpreferably, but not necessarily, occurs under substantially the sameconditions at each location on the array.

[0026] The present invention is suitable for the detection of anynucleic acid. For example, if a sample were suspected of containing anucleic acid specific for a pathogen, the sample could be analyzed bythe present invention. A novel primer would be synthesized with a 3′portion specific for the pathogen genome and a 5′ portion complementaryto a preselected sequence. The sample would be either analyzed directlyor after amplification by PCR.

[0027] The present invention is also suitable for discriminating betweenindividuals on the basis of genetic differences. For example, thedetermination of which alleles are present at 20 different dimorphic,genetically unlinked loci would provide a powerful method useful inforensic science to discriminate between different individuals. Thepresent invention is useful, e.g., in transplantation medicines, todetermine which HLA alleles are present in a DNA sample of anindividual.

EXEMPLIFICATION OF THE INVENTION EXAMPLE I

[0028] Experimental Design

[0029] The first step, not shown, involves the amplification of thetarget sequence with a primer set (TYR 1 and TYR 2) specific for the TYRlocus. After the amplification, the template is prepared for the nextstep by eliminating the remaining dNTPs.

[0030]FIG. 4 shows an overview of the procedure called ASPE-capture,applied in this example to the detection of the amplified dimorphicsequence present at the TYR locus produced by the first step.

[0031] The method consists of three steps. This procedure is locus andallele specific in that it is designed both to specifically detect thepresence of the amplification product and to determine the targetidentifying polymorphic nucleotide of the analyzed locus. FIG. 4 depictstwo separate primer extension reactions which have as common ingredientsthe DNA template, the ASPE primer, and the Ampli-Taq polymerase. The tworeactions differ in that they contain different nucleosidetriphosphates: allele A1 is detected by including α-³²p dGTP and alleleA2 by including α-³²p TTP. The ASPE primer is a 40 nucleotide longoligonucleotide which includes two different portions (X and Y) eachhaving a different role. Sequence Y is identical to the sense sequenceof the TYR gene with its 3′ nucleotide immediately flanking thepolymorphic base. This portion of the ASPE primer participates in theprimer extension reaction. Y sequence is extended by the Ampli-Taq DNApolymerase whenever an α-³²p labeled nucleotide complementary to thepolymorphic base is present in the reaction. For a DNA template fromindividuals homozygous for the TYR-A1 allele, the ASPE primer will beonly extended by α-³²p labeled dGTP. α-³²p labeled TTP will be added tothe ASPE primer when a DNA template from individuals homozygous for theTYR-A2 allele is analyzed. Both labeled nucleoside triphosphates will beadded to the ASPE primer for DNA templates from heterozygousindividuals. It should be noted that if no template is present, nolabeling of the ASPE primer will happen. Sequence X is complementary toa grid oligonucleotide, thus permitting capture of the primer extensionproduct on the grid.

[0032] This invention is not limited to the described steps. Forexample, the labeling of the primer extension product could beaccomplished using fluorescently labeled deoxynucleoside triphosphatesor dideoxynucleoside triphosphates. The capture could be accomplished byhybridization in solution to an oligonucleotide which can later beimmobilized (for example by utilizing biotin-avidin, hapten-antibodyreactions).

EXAMPLE II

[0033] Synthesis of Oligonucleotides

[0034] Chemical synthesis of oligonucleotide primers

[0035] Oligonucleotides were synthesized on a Cruachem PS 250 DNAsynthesizer. Oligonucleotides for amplifying the target sequence (TYR 1and TYR 2) were purified by HPLC. The oligonucleotide used in the allelespecific primer extension reactions (ASPE primer) was purified by MPLCfollowed by a 20% polyacrylamide gel/7M urea. The sequences of thesynthetic oligonucleotides utilized are presented in Table 1. TABLE 1Oligonucleotide Sequences Specific For TYR TYR 1 5′GCAAGTTTGGCTTTTGGGGATYR 2 5′CTGCCAAGAGGAGAAGAATG ASPE5′TGACGTCCATCGTCTCTGCGAATGTCTCTCCAGATTTCA primer

[0036] Chemical synthesis of grid oligonucleotide

[0037] The grid oligonucleotide for attachment to the nylon membrane wasprepared as follows: 3′Amine-ON CPC columns (0.2 μmole) were purchasedfrom Cruachem and were used on the Cruachem PS 250 DNA synthesizer.Three 1,3 propanediol nucleoside substitutes were coupled directly tothe column prior to coupling the grid oligonucleotide sequence. Theprotected 1,3 propanediol phosphoramidite was prepared essentially asdescribed by Seela and Kaiser, Nucleic Acids Res. 15:3113-3129 (1987).This phosphoramidite was placed in the X position of the synthesizer.For example to synthesize the grid oligonucleotide for the detection ofTYR extension products, the synthesizer was programmed to synthesize5′CGCAGAGACGATGGACGTCAXXX. The oligonucleotide is deprotected as usualand the oligonucleotide recovered by ethanol precipitation. Theoligonucleotide contains a 3′NH₂ group.

[0038] Preparation of the filters containing grid oligonucleotides

[0039] The membranes containing the grid oligonucleotides were preparedaccording to Zhang, et al., Nucleic Acids Res. 19:3929-3933 (1991).Briefly, Biodyne C membranes (Pall Biosupport, N.J.) were rinsed with0.1N HCl and then treated for 15 minutes with 20% EDC(1-Ethyl-3-[di-methylamion propyl] carbodiimide hydrochloride) (w/v) indeionized water. After the activation process, the membranes wereimmediately set in a 96 well Bio-dot apparatus (Bio-Rad, Richmond,Calif.). The grid oligonucleotide was mixed with 0.5 M sodiumbicarbonate buffer pH 8.4 and applied to the membranes for 15 minutes.The membranes were rinsed with TBS/0.1% Tween-20 (Tris buffered saline)and then treated with 0.1 N NaOH for 10 minutes. Finally, filters wererinsed with deionized water.

EXAMPLE III

[0040] Allele Specific Detection Reactions

[0041] Isolation of genomic DNA:

[0042] Blood samples were collected from ten unrelated individuals. Highmolecular weight DNA was prepared according to a modified procedureusing Triton X-100 followed by Proteinase K and RNAse treatment (Bell,et al., Proc. Natl. Acad. Sci. USA 78:5759-5763 (1981)).

[0043] In vitro amplification of DNA template:

[0044] DNA (50 ng) from ten random individuals was amplified with aprimer set derived from exon 1 of the human tyrosinase gene (TYR)(GenBank Accession M27160; Locus HUMTYRA) in a 50 μl reaction volumecontaining 50 mM KC1, 10 mM Tris—HCl (pH 8.3), 1.5 mM MgCl₂, 0.01% (w/v)gelatin, 10 pmol of each primer, 0.2 mM each DATP, dCTP, dGTP, TTP, and2.5 units of Ampli-taq polymerase (Perkin Elmer Cetus). The reaction wasperformed in a thermal cycler (Perkin Elmer). The reaction mixture wasdenatured at 94° C. for 3 minutes, then the cycle was continued byannealing the primers at 65° C. for 1 minute and extending the primersat 72° C. for 1 minute. After extension, the samples were denatured at94° C. for 30 seconds. The annealing, extension, and denaturation cyclewas repeated 40 times. After the last cycle, the samples were incubatedat 65° C. for 1 minute, at 72° C. for 6 minutes and finally at 4° C.until analyzed.

[0045] Preparation of DNA template for primer extension reactions:

[0046] In order to control the quality of the PCR products, 5 μl wereloaded in a 1.5% agarose gel. The remaining 45 μl were ethanolprecipitated twice using 2.5 M ammonium acetate and 10 μg of glycogen.The pellet was resuspended in H₂O (100 μl).

[0047] Allele-specific primers extension reactions:

[0048] Each allele-specific primer extension reaction (ASPE) wereperformed in a 10 μl volume containing 1 μl of PCR template, 1 μl of 10×PCR buffer (1×=10 mM Tris—HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl2), 0.01%(w/v) gelatin, 0.25 μM of ASPE primer, 1 unit of Ampli-taq polymerase,and 0.5 μl of the appropriate α-³²P-labeled nucleotide (10 μCi/μl, 3000Ci/mmol). To analyze the dimorphism present at locus TYR, each samplewas subjected to two separate primer extension reactions using α-³²Plabeled dGTP and TTP respectively. Mixtures were denatured at 94° C. for3 minutes and then subjected to 10 cycles consisting of 1 secondannealing at 55° C. and 30 seconds denaturation at 94° C. After thereaction, samples were ethanol precipitated and used for hybridization.

[0049] Hybridization and post hybridization washes:

[0050] All the hybridizations were performed overnight at 55° C. in 2 mlmicro centrifuge tubes using hybridization incubator (RobbinsScientific, model 310). The hybridizations were done in a 225 μl volume(Hybridization solution: 5× SSPE [1× SSPE=10 mM sodium phosphate pH 7.0,0.18 M NaCl and 1 mM EDTA], 1% SDS, 0.5% (w/v) dehydrated powdered skimmilk (Carnation, Los Angeles, Calif.), 10 μg/ml homomix RNA, and theproduct of the primer extension reaction). After hybridization, thefilters were washed with 6× SSC (1× SSC=0.15 M NaCl, 0.015 M sodiumcitrate) at room temperature for 15 minutes. Autoradiography ofmembranes was done using Kodak X-AR5 film and exposing for 15-30 minutesat room temperature. Subsequently, the amount of cpm corresponding tothe labelled ASPE bound to the filter was measured by Ambis-scan (AmbisCorp., San Diego, Calif.).

[0051] The results of hybridization are shown in FIG. 5. Note that ofthe ten individuals analyzed, six are heterozygous (2, 3, 4, 5, 6 and10) and four are homozygous for the TYR-A1 allele.

EXAMPLE IV

[0052] Grid Oligonucleotides

[0053] General approach for the design of grid design of gridoligonucleotide sequences

[0054] Grid oligonucleotides were designed to be 20 nucleotides inlength and to have a base composition of 50% G+C. This design allowshybridization reactions to be carried out with a single hybridizationtemperature for all sequences The first 20 grid oligonucleotidesequences were generated by starting with the sequence5′GGGGGCCCCCTTTTTAAAAA (25% of each of the four bases). This sequencewas then randomized by the RANDOMIZATION option (group length 1,randomize all) within the program GENED in the Intellegenetic Suitesoftware for molecular biology (Intelligenetics, Mountain View, Calif.)to give the first sequence in Table 2. Each of the next 19 sequenceswere generated by randomizing the previous sequence in the list. TABLE 2Examples of Grid Oligonucleotide Sequences 5′TCGCGTTGCATAAGATCCGA5′CTTAACGAAAGCTGCGGTCT 5′TTCGAGCTCCAGTTAACGAG 5′TCTCTATGTCGGAAGAGCCA5′CATAATGCGGTCTCGATACG 5′ATTGCCGTCCGTAGGACTAA 5′TCTATCACGGACTATCGGGA5′TTGTCACAGGCACAATGTGC 5′CGAAGAGCCATGATGCTTCT 5′AGACGTCGTCACGATTCTGA5′ACACGTGCGCCTGGAATTAT 5′CAGGTGCCATATAATGGTCC 5′TGGCCATACCAGACTTTAGG5′ATGGGCTCCTGCGTAAATCA 5′AGTGCGCTCTCTTGAGCAAA 5′ACTGTTACCGGTAACTGACG5′GATATACCATTCCAGGCGTG 5′TCCGGCTCATGGTAGAATAC 5′CTTACAATATCCGGCGTGGA5′GGGTATTCTCGACCAATCAG

[0055] Similarly, additional grid oligonucleotides can be generatedstarting with other sequences which are 50% G-C (e.g.,5′AAATTTTTTTGGGCCCCCCC, 5′GGGGGGGCCCAAAAAATTTT).

EXAMPLE V

[0056] Removal of Deoxynucleoside Triphosphates

[0057] An important step in the practice of this invention is theremoval of the deoxynucleoside triphosphates (dNTPs), if present, fromthe sample to be subjected to the primer extension reaction. This can beaccomplished by several methods including ethanol precipitation of thetemplate DNA (as described in Example III), by destruction of the dNTPswith an enzyme or by chromatography on a column which can separatepolymerized nucleic-acids from dNTPs.

[0058] In the latter approach, a column of DE52 was prepared. DE52(Whatman) was suspended in a buffer containing 10 mM Tris—HCl, pH 7.5(TE) such that the ratio of DE52 to buffer was 1:2. The DE52 suspension(0.2 ml) was poured into a 1 ml micropipette tip (e.g., the blue tip forGilson P-1000 Pipetman™) which had been plugged with glass wool andwashed with 1 ml of TE. The sample (e.g., the product of a polymerasechain reaction) was diluted to 0.1 ml with TE and applied to the columnunder slight air pressure. The column was washed with 1 ml of TE. ThedNTPs were eluted with 4×0.5 ml of 0.2 M NaCl. Finally, the sample wasrecovered, essentially free of dNTPs by 0.2 ml 1M NaCl. The sample couldthen be used directly in a primer extension reaction.

1 27 20 Nucleotide Single Linear 1 GCAAGTTTGG CTTTTGGGGA 20 20Nucleotide Single Linear 2 CTGCCAAGAG GAGAAGAATG 20 39 Nucleotide SingleLinear 3 TGACGTCCAT CGTCTCTGCG AATGTCTCTC CAGATTTCA 39 20 NucleotideSingle Linear 4 TCGCGTTGCA TAAGATCCGA 20 20 Nucleotide Single Linear 5CTTAACGAAA GCTGCGGTCT 20 20 Nucleotide Single Linear 6 TTCGAGCTCCAGTTAACGAG 20 20 Nucleotide Single Linear 7 TCTCTATGTC GGAAGAGCCA 20 20Nucleotide Single Linear 8 CATAATGCGG TCTCGATACG 20 20 Nucleotide SingleLinear 9 ATTGCCGTCC GTAGGACTAA 20 20 Nucleotide Single Linear 10TCTATCACGG ACTATCGGGA 20 20 Nucleotide Single Linear 11 TTGTCACAGGCACAATGTGC 20 20 Nucleotide Single Linear 12 CGAAGAGCCA TGATGCTTCT 20 20Nucleotide Single Linear 13 AGACGTCGTC ACGATTCTGA 20 20 NucleotideSingle Linear 14 ACACGTGCGC CTGGAATTAT 20 20 Nucleotide Single Linear 15CAGGTGCCAT ATAATGGTCC 20 20 Nucleotide Single Linear 16 TGGCCATACCAGACTTTAGG 20 20 Nucleotide Single Linear 17 ATGGGCTCCT GCGTAAATCA 20 20Nucleotide Single Linear 18 AGTGCGCTCT CTTGAGCAAA 20 20 NucleotideSingle Linear 19 ACTGTTACCG GTAACTGACG 20 20 Nucleotide Single Linear 20GATATACCAT TCCAGGCGTG 20 20 Nucleotide Single Linear 21 TCCGGCTCATGGTAGAATAC 20 20 Nucleotide Single Linear 22 CTTACAATAT CCGGCGTGGA 20 20Nucleotide Single Linear 23 GGGTATTCTC GACCAATCAG 20 20 NucleotideSingle Linear 24 AAATTTTTTT GGGCCCCCCC 20 20 Nucleotide Single Linear 25GGGGGGGCCC AAAAAATTTT 20 23 Nucleotide Single Linear 26 CGCAGAGACGATGGACGTCA NNN 23 20 Nucleotide Single Linear 27 GGGGGCCCCC TTTTTAAAAA20

1. A primer having a 3′ portion complementary to a target nucleic acidsequence adjacent to at least one specific nucleotide which may or maynot be present in a sample and a 5′ portion complementary to apreselected nucleotide sequence.
 2. A method for detecting the presenceor absence of a target nucleic acid sequence in a sample, said methodcomprising (i) adding to said sample an oligonucleotide primer toprovide a primer extension product including said target sequence, ifpresent, in said sample, said primer having a 3, end portioncomplementary to a portion of said target sequence immediately adjacentat least one specific nucleotide to provide an extension product of saidprimer which includes said specific nucleotide, and a 5′ end portioncomplementary to a preselected nucleic acid sequence different from saidtarget sequence; (ii) adding to the product of step (i) a DNA polymeraseand one or more labeled deoxynucleoside triphosphates to provide alabeled extension product of said primer which includes said targetsequence and said specific nucleotide if present; (iii) subjecting saidlabeled primer extension product to hybridization conditions with apreselected nucleic acid sequence complementary to the 5′ end portion ofsaid primer; and (iv) determining whether hybridization occurs.
 3. Themethod according to claim 2 wherein said sample may contain a pluralityof target sequences and wherein said sample is subjected to polymerasechain reaction amplification with at least one pair of oligonucleotideprimers for two or more of said target sequences.
 4. The methodaccording to claim 2 wherein said nucleic acid target sequence$ arealleles of one another, the 3′ portion of said primer is positionedimmediately adjacent to the variant nucleotide responsible for theallelism, and said primer extension reactions are independentlyperformed with each of the four deoxynucleoside triphosphates such thatonly one primer extension reaction will occur for each of said alleles,said deoxynucleoside triphosphate being labeled whereby a single labeledprimer extension product is produced-for each of said alleles.